Cell Wall Layer Induced in Xylem Fibers of Flax Upon Gravistimulation Is Similar to Constitutively Formed Cell Walls of Bast Fibers

Abstract

In the fibers of many plant species after the formation of secondary cell walls, cellulose-enriched cell wall layers (often named G-layers or tertiary cell walls) are deposited which are important in many physiological situations. Flax (Linum usitatissimum L.) phloem fibers constitutively develop tertiary cell walls during normal plant growth. During the gravitropic response after plant inclination, the deposition of a cellulose-enriched cell wall layer is induced in xylem fibers on one side of the stem, providing a system similar to that of tension wood in angiosperm trees. Atomic force microscopy (AFM), immunochemistry, and transcriptomic analyses demonstrated that the G-layer induced in flax xylem fibers was similar to the constitutively formed tertiary cell wall of bast (phloem) fibers but different from the secondary cell wall. The tertiary cell walls, independent of tissue of origin and inducibility, were twice as stiff as the secondary cell walls. In the gravitropic response, the tertiary cell wall deposition rate in xylem was higher than that of the secondary cell wall. Rhamnogalacturonan I (RG-I) with galactan side chains was a prominent component in cellulose-rich layers of both phloem and xylem flax fibers. Transcriptomic events underlying G-layer deposition in phloem and xylem fibers had much in common. At the induction of tertiary cell wall deposition, several genes for rhamnosyltransferases of the GT106 family were activated in xylem samples. The same genes were expressed in the isolated phloem fibers depositing the tertiary cell wall. The comparison of transcriptomes in fibers with both inducible and constitutive tertiary cell wall deposition and xylem tissues that formed the secondary cell walls is an effective system that revealed important molecular players involved in the formation of cellulose-enriched cell walls.

Keywords: atomic force microscopy; biomechanical properties; flax fibers; gravitropic response; rhamnogalacturonan I; rhamnosyltransferases; tertiary cell wall; transcriptome.

FIGURE 1

Schematic of the experimental design. Flax plants were inclined and stapled to the soil at the level of the cotyledons. Gravitropic response developed over 96 h. Five-cm-long stem segments beginning at 3 cm above the cotyledons were used in the analyses. The upper part of the gravitropic bend was designated as the pulling side (PUL) and the lower part as the opposite side (OPP). Phloem and xylem (XYL) parts of the stem were collected separately for RNA-Seq in the control and after 8, 24, and 96 h of gravitropic response. Phloem parts were washed several times in ethanol in a mortar with gentle pestling to isolate phloem fibers (FIB). All samples for fluorescence microscopy and atomic force microscopy were collected 96 h after gravistimulation. Control samples were taken from the non-inclined plants, at the same stem level and at the same time as for the inclined plants.

FIGURE 2

Fluorescence micrographs of flax stem cross sections immunolabeled by (A) LM11 (xylan) and (B) LM5 (1,4-galactan) antibodies in control plants and after 4 days (96 h) of gravistimulation. Bars are 100 μm. Ph, phloem; X, xylem. (C) Mean fluorescence intensity of different cell walls. TCW, tertiary cell wall; G and Gn, G and Gn layers. Values are the mean ± SD. Different letters above the bars indicate a significant difference according to one-way ANOVA followed by Tukey’s test at α = 0.01.

FIGURE 3

Atomic force microscopy of fiber cell walls in control and gravistimulated flax plants 96 h after stem inclination. (A) Stiffness (N/m) maps of phloem (top) and xylem (bottom) fibers in control and gravistimulated flax plants. One representative image for each variant is shown. Stiffness scales are on the right side of each map. Bars are 10 μm. G- and Gn-layers of tertiary cell walls in phloem and similar structures in the cell walls of xylem fibers on the pulling side are indicated. S indicates secondary cell walls. Parallel lines on each image are scratches left by the knife. (B) Thickness (μm) and (C) Apparent elasticity modulus (GPa) of different cell wall layers. SCW, secondary cell walls; TCW, tertiary cell walls. Values are the mean ± SD. Different letters above the bars indicate a significant difference according to one-way ANOVA followed by Tukey’s test at α = 0.01 (individual comparisons are shown by regular, italic, and bold-type fonts of these letters).

FIGURE 4

Maximum-likelihood phylogenetic dendrogram of plant GT106 protein family members (with PF10250 and IPR024709 domains) of flax (black) and Arabidopsis thaliana (red). Dark gray confines the RG-I:rhamnosyltransferase (RRT) clade, according to Takenaka et al. (2018), and light gray indicates the RRT clade size, according to Wachananawat et al. (2020). Purple arrows show genes upregulated in the pulling side xylem during gravitropic response. The A. thaliana gene names are according to the annotation of the Uniprot database⁸ (Bateman et al., 2019). Numbers indicate the ultrafast bootstrap support values; branch lengths were ignored.